Construction of porous chitosan macrospheres via dual pore-forming strategy as host for alkaline protease immobilization with high activity and stability

Fabrication of highly-efficient enzymatic supports having excellent affinity to enzymes and superior mass transfer properties is highly desirable for enzymatic bio-catalysis. Herein, newly engineered chitosan macrospheres having interconnected and interlaced network pores are prepared via dual pore-forming strategy and applied as novel host for the effective immobilization of alkaline protease. The synergetic effect of SiO2 templates and gas-induced pore-forming agents play an important role in inhibiting the over-crosslinking of chitosan chains and promoting the elevation of interior porosity. Benefited from the highly exposed surface and abundant available binding sites, the as-developed porous support P2CSM achieves a maximum loading capacity of 43.8 ± 0.8 mg/g and ultra-high activity recovery of 92.4 % for alkaline protease. P2CSM is competent to effectively stabilize the structural conformation of alkaline protease from inactivation through the flexible covalent interaction. Considering these attributes, Protease@P2CSM demonstrates remarkably better structural stability, reusability and SDS-resistance than free alkaline protease, as well as excellent proteolytic ability, and the residual activity of Protease@P2CSM is evaluated as high as 70.3 % after 7 consecutive reuses. This work provides a promising avenue to construct highly-active enzyme-composites for widespread utilization in various practical applications.